CN103194524B - Method for rapidly identifying peanut germplasm with bacterial wilt resistance - Google Patents
Method for rapidly identifying peanut germplasm with bacterial wilt resistance Download PDFInfo
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- CN103194524B CN103194524B CN201310116635.3A CN201310116635A CN103194524B CN 103194524 B CN103194524 B CN 103194524B CN 201310116635 A CN201310116635 A CN 201310116635A CN 103194524 B CN103194524 B CN 103194524B
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Abstract
The invention relates to a plant germplasm identification technology and discloses a method for rapidly identifying peanut germplasm with bacterial wilt resistance. The method comprises the following steps of: (1) culturing ralstonia solanacearum of peanuts and preparing bacterium suspension with concentration of 108p/ml or 109p/ml; (2) cutting stems and branches with leaves from the top ends of the second pair of side branches of healthy peanut plants growing 55-65 days after sowing, and culturing the cut stems and branches in the bacterium suspension; and (3) after the stems and branches are cultured for 3 days, judging whether the peanut plants resist bacterial wilt from the wilt degrees of the measured stems and branches, wherein the peanut plants without obvious wilt symptoms in the bacterium suspension with concentration of 108p/ml are the peanut germplasm with bacterial wilt resistance, and the peanut plants without obvious wilt symptoms in the bacterium suspension with concentration of 109p/ml are the peanut germplasm with high bacterial wilt resistance. The plant germplasm identification technology has the advantage of overcoming the defects that an existing identification method for the bacterial wilt resistance of peanuts is long in period and fussy in operation, is difficult to identify the resistance of the material with segregative generation and is disjointed with genetic breeding practice due to no influence on selection of other agronomic traits.
Description
Technical field
The present invention relates to plant germplasm authenticate technology, be specifically related to a kind of method obtaining Peanut Genotypes with Bacterial Wilt Resistance.
Background technology
Peanut is one of five kinds of most important oil crops in the world, is also the important sources of many developing countries vegetables oil and protein.China is maximum in the world peanut production, consumption and export State.The bacterial wilt (bacterial wilt) caused by Ralstonia solanacearum is one of disease important in peanut production, often causes dropping in production over a large area and even has no harvest.As soil-borne disease, bacterial wilt of peanut is difficult to chemically prevent and treat, and the crop rotation of longer cycle has certain effect, but it limits by cultivated area.Biological control is also a kind of important method, and obtain certain effect, Chinese patent application disclosed in 28 days July in 2004, its publication number is CN1515666A, disclose a kind of screening method for preventing and treating bacterial wilt pseudomonas, but because bacterial wilt of peanut all has generation at peanut each period (seedling stage, initial bloom stage, full-bloom stage etc.), cause biological control costly, so cultivating and plant disease-resistant variety is the comparatively cost-effective approach of this disease of control.
Silk channel injection Resistance to bacterial wilt can in field and indoor qualification.Natural infection method needs the ground of disease naturally that soil fertility is even, physical features is smooth, Disease pressure is consistent, and can only carry out season at peanut growth, the cycle is longer, adds up plant survival rate, be difficult to the disease resistance determining individuality (single seed or single plant) during results.Artificial infection idenfication method, seed can be soaked in bacterium liquid and then be seeded in field, other step is identical with Natural infection method; Or when water planting peanut growth is to 3-4 leaf age, employing is hindered root leaching root method and is carried out Ralstonia solanacearum inoculation process to peanut, 10-15 days " Invest, Then Investigate " plant incidences; Also the plant can cultivated in flowerpot carries out leaf-cutting inoculation: in the peanut 6 leaf phase, second and third two panels leaflet 1/4 place, leaf top shearing blade, often dips in 1 bacterium liquid and cuts 2 leaves from the top down.Leaf-cutting plant is placed in 26 DEG C of incubators, and humidity records incidence after remaining 75%, 4d.Wherein latter two method can be used for the resistance determining single plant.But still existence takies larger space, is difficult to put into practice with genetic breeding the problem combined.Therefore, in the urgent need to one by the time, space constraint is less, Rapid identification can obtain the method for anti-Ralstonia solanacearum Silk channel injection in the lab.
Summary of the invention
Technique effect of the present invention can overcome above-mentioned defect, and provide a kind of method of Rapid identification Peanut Genotypes with Bacterial Wilt Resistance, it carries out in field plant process of growth, can in the technology of the Rapid identification Peanut Genotypes with Bacterial Wilt Resistance of experiment in-house operation.
For achieving the above object, the present invention adopts following technical scheme: it comprises the steps:
(1) peanut Ralstonia solanacearum is cultivated, preparation 10
8individual/ml or 10
9the bacteria suspension of individual/ml concentration;
(2) cut long vaned stem branch from healthy peanut plant the 2nd pair of side shoot top broadcasting rear 55-65 days, be placed in above-mentioned bacteria suspension and cultivate;
(3) cultivate after 3 days, judging this strain peanut whether resistance to bacterial wilt from the withered degree of tested stem branch, is 10 in concentration
8in the bacterium liquid of individual/ml, what do not occur remarkable wilting symptoms is Peanut Genotypes with Bacterial Wilt Resistance; Be 10 in concentration
9in the bacterium liquid of individual/ml, what do not occur remarkable wilting symptoms is high Peanut Genotypes with Bacterial Wilt Resistance.
Content of the present invention is that from peanut plant, part stem branch is got on the 2nd pair of side shoot top, and being placed in concentration is 10 in peanut growth period
8individual/ml or 10
9dual culture in the peanut Ralstonia solanacearum bacteria suspension of individual/ml, after cultivating after a while, judges its Resistance to bacterial wilt from the change of stem branches and leaves sheet.
Ralstonia solanacearum in step (1) is streak culture on NA solid medium.NA solid medium component is that pH value is 7.0-7.2 containing glucose 10g, peptone 5g, extractum carnis 3g, yeast powder 0.5g, agar 20g in often liter of substratum.This substratum is published Ralstonia solanacearum minimum medium.
Ralstonia solanacearum training method in step (1) is: picking list bacterium colony is uniformly coated on NA solid medium, covers with after media surface, rinsed with distilled water until lawn, calculate its concentration, bacterial concentration is adjusted to 10 with blood counting chamber after suspending
8individual/ml or 10
9individual/ml.
When cutting in step (2), blade becomes vertical direction to cut with stem branch, and sectional area is minimized, and reduces being cut the infringement of branch, can make to be cut contacting with bacterium liquid of place's homogeneous microstructure, improves experiment accuracy.
Culturing step in step (2) is placed in illumination box, and 28 DEG C by photoperiod and alternate culture of each 12 hours of dark cycle.This condition imitates the self-sow environment of field peanut at bacterial wilt occurrent time, and this temperature is the optimum growth temperature of Ralstonia solanacearum, is conducive to accurately recording peanut to be measured under bacterium liquid-stem branch Dual culture condition and whether has Resistance to bacterial wilt.
The technology of the present invention compensate for existing bacterial wilt of peanut Resistance Identification method cycle length, complex operation, is difficult to identify that segregating generation material resistance does not affect other economical character simultaneously and selects and put into practice with genetic breeding the deficiency disconnected.
Embodiment
The method of Rapid identification Peanut Genotypes with Bacterial Wilt Resistance of the present invention, comprises the steps:
(1), by streak culture on NA substratum for the Ralstonia solanacearum preserved, after picking list bacterium colony suspends, coating NA substratum, covers with after media surface until lawn, is rinsed with distilled water.Calculate its concentration with blood counting chamber, bacterial concentration is adjusted to 10
8individual/ml or 10
9individual/ml.
(2), before in the morning 10 time, cut the terminal bud of after planting about 60 days healthy peanut plants the 2nd pair of side shoot, when cutting, blade becomes vertical direction with stem, cut rapidly, be placed in above-mentioned bacteria suspension immediately, put in 28 DEG C of illumination boxs, cultivate by photoperiod and each 12 hours of dark cycle.
(3) cultivate after 3 days, observing stem branch and to wither situation, is 10 in concentration
8in the bacterium liquid of individual/ml, what do not occur remarkable wilting symptoms is Peanut Genotypes with Bacterial Wilt Resistance (comprise high resistance and in anti-); Be 10 in concentration
9in the bacterium liquid of individual/ml, what do not occur remarkable wilting symptoms is high Peanut Genotypes with Bacterial Wilt Resistance.
We identify the 40 parts of peanut samples preserved Shandong Peanut Inst.'s Biotechnology Experiment room by method of the present invention, learn its resistance to bacterial wilt characteristic by field experiment before this batch sample.These 40 increment product are sowed, healthy and strong stem branch is cut when it grows the 2nd pair of side shoot, be placed in the bacteria suspension Dual culture prepared by method of the present invention, experiment terminates rear statistics, and the result that the Resistance to bacterial wilt qualification result of all 40 parts of peanut samples and field experiment draw is completely the same.The withered degree of Resistant gerplasm and susceptible kind matter presents marked difference.
Claims (6)
1. a method for Rapid identification Peanut Genotypes with Bacterial Wilt Resistance, is characterized in that, comprises the steps:
(1) peanut Ralstonia solanacearum is cultivated, preparation 10
8individual/ml or 10
9the bacteria suspension of individual/ml concentration;
(2) cut long vaned stem branch from healthy peanut plant the 2nd pair of side shoot top broadcasting rear 55-65 days, be placed in above-mentioned bacteria suspension and cultivate;
(3) cultivate after 3 days, judging this strain peanut whether resistance to bacterial wilt from the withered degree of tested stem branch, is 10 in concentration
8in the bacterium liquid of individual/ml, what do not occur remarkable wilting symptoms is Peanut Genotypes with Bacterial Wilt Resistance; Be 10 in concentration
9in the bacterium liquid of individual/ml, what do not occur remarkable wilting symptoms is high Peanut Genotypes with Bacterial Wilt Resistance.
2. the method for Rapid identification Peanut Genotypes with Bacterial Wilt Resistance according to claim 1, is characterized in that, the Ralstonia solanacearum in step (1) is streak culture on NA solid medium.
3. the method for Rapid identification Peanut Genotypes with Bacterial Wilt Resistance according to claim 2, it is characterized in that, NA solid medium component is that pH value is 7.0-7.2 containing glucose 10g, peptone 5g, extractum carnis 3g, yeast powder 0.5g, agar 20g in often liter of substratum.
4. the method for Rapid identification Peanut Genotypes with Bacterial Wilt Resistance according to claim 2, it is characterized in that, Ralstonia solanacearum training method in step (1) is: after picking list bacterium colony suspends, be uniformly coated on NA solid medium, cover with after media surface until lawn, rinsed with distilled water, calculate its concentration with blood counting chamber, bacterial concentration is adjusted to 10
8individual/ml or 10
9individual/ml.
5. the method for Rapid identification Peanut Genotypes with Bacterial Wilt Resistance according to claim 1, is characterized in that, when cutting in step (2), blade becomes vertical direction to cut with stem branch.
6. the method for Rapid identification Peanut Genotypes with Bacterial Wilt Resistance according to claim 1 or 5, it is characterized in that, the culturing step in step (2) is placed in illumination box, and 28 DEG C by photoperiod and alternate culture of each 12 hours of dark cycle.
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112111550A (en) * | 2020-08-26 | 2020-12-22 | 山东省花生研究所 | Identification method for grading resistance to peanut bacterial wilt and application of identification method |
| CN112237134A (en) * | 2020-09-03 | 2021-01-19 | 广东省农业科学院蚕业与农产品加工研究所 | Rapid breeding method of bacterial wilt-resistant mulberry variety |
| CN113667712A (en) * | 2021-09-02 | 2021-11-19 | 百色学院 | Artificial inoculation method for plant bacterial vascular bundle diseases |
| CN115474487A (en) * | 2022-08-30 | 2022-12-16 | 仲恺农业工程学院 | Indoor ralstonia solanacearum inoculation method |
| CN115976151A (en) * | 2022-12-12 | 2023-04-18 | 中国农业科学院油料作物研究所 | Water culture inoculation method for rapidly and efficiently identifying peanut bacterial wilt resistance |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102021223A (en) * | 2009-09-17 | 2011-04-20 | 中国农业科学院棉花研究所 | Quantitative dip in bacterial suspension method for identifying resistance to cotton verticillium wilt using vermiculite sandy soil bottomless bowl of paper |
| CN102220407A (en) * | 2011-05-12 | 2011-10-19 | 云南农业大学 | Method for identifying gray mold resistance of gerbera |
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| EP0866129A2 (en) * | 1996-12-31 | 1998-09-23 | Novartis AG | Genomic DNA sequences of Ashbya gossypii and uses thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102021223A (en) * | 2009-09-17 | 2011-04-20 | 中国农业科学院棉花研究所 | Quantitative dip in bacterial suspension method for identifying resistance to cotton verticillium wilt using vermiculite sandy soil bottomless bowl of paper |
| CN102220407A (en) * | 2011-05-12 | 2011-10-19 | 云南农业大学 | Method for identifying gray mold resistance of gerbera |
Non-Patent Citations (1)
| Title |
|---|
| 花生青枯病的发生与抗性研究;曾东方等;《湖北农业科学增刊》;19940130(第1期);第74页第1段、材料和方法部分、结果和分析部分，第75页左栏第1段、右栏第1段、讨论部分、图1、表2 * |
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